5-Bromo-1-(2-deoxy-β-d -ribofuranosyl) uracil (5-BrdU, CAS: 59-14-3), a fixing/denaturing solution (FixDenat, Cat# 11758764001), anti-BrdU- peroxidase (POD) from mouse IgG1_clone BMG6H8 (Cat# 11585860001), 2-methyl-4-isothiazolin-3-one hydrochloride (CAS: 26172-54-3), lead(II) sulfide (PBS, CAS: 1314-87-0), 3,3′,5,5′-tetramethylbenzidine (TMB, CAS: 54827-17-7), diaphorase (CAS: 9001-18-7); Triton X-100 (CAS: 9002-93-1), and 2,3,5-triphenyltetrazolium chloride (CAS: 146-68-9) were purchased from Roche (Sigma-Aldrich), Milan, Italy. 6-Hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid (Trolox, CAS: 53188-07-1), 2,2′-diphenyl-1-picrylhydrazyl (DPPH, CAS: 1898-66-4), 2,2′-azino-bis-(3-etilbenzotiazolino-6-sulfonic acid) (ABTS, CAS: 30931-67-0), potassium persulfate (CAS: 7727-21-1), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, CAS: 298-93-1), and 2-propanol were purchased from Sigma-Aldrich (Milan, Italy). Roswell Park Memorial Institute medium (RPMI) 1640, Dulbecco’s modified Eagle’s medium (DMEM, with phenol red and phenol red-free), Dulbecco’s phosphate-buffered saline (DPBS), l -glutamine, trypsin–EDTA, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), penicillin/streptomycin (10,000 U/mL), foetal bovine serum (FBS), and nonessential amino acids (MEM, 100×) were purchased from Lonza Bio Whittaker (Verviers, Belgium).
3 3 5 5 tetramethylbenzidine (tmb)
Manufactured by Roche
Sourced in Germany
TMB is a chromogenic substrate used in enzyme-linked immunosorbent assays (ELISA) to detect and quantify the presence of specific analytes in a sample. It undergoes a color change upon oxidation by the enzyme horseradish peroxidase (HRP), which is commonly conjugated to antibodies or other detection reagents in ELISA protocols. The intensity of the resulting color change is proportional to the amount of the target analyte in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Rpn4201, by Cytiva (2 mentions)
Prism 6, by GraphPad (2 mentions)
Donkey f ab 2 anti rabbit igg pod, by Cytiva (2 mentions)
Potassium persulfate, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Fixdenat, by Roche (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
6 hydroxy 2 5 7 8 tetramethylchromane 2 carboxylic acid trolox, by Merck Group (1 mentions)
2 propanol, by Merck Group (1 mentions)
Triton x 100, by Roche (1 mentions)
Brdu labeling solution, by Roche (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Model 550, by Bio-Rad (1 mentions)
Hrp conjugated goat anti mouse igg, by Merck Group (1 mentions)
Goat anti rabbit igg hrp conjugate, by Merck Group (1 mentions)
Na9340v, by Cytiva (1 mentions)
96 well culture plate, by Thermo Fisher Scientific (1 mentions)
Streptavidin hrp, by Roche (1 mentions)
9 protocols using 3 3 5 5 tetramethylbenzidine (tmb)
1
Standardized Cell Viability Assay Protocol
2
Screening for Anti-AG-GMBS-BSA Antibodies
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ELISA was performed similarly to our previous method for anti-DM mAbs [11 (link)]. For screening clones producing antibody against AG-GMBS-BSA, the wells in microtiter plates were coated with 100 μl of the AG-GMBS-BSA conjugate (10 μg/ml) for 60 min at 37°C and then blocked the protein binding sites with 1% skimmed milk for 30 min at room temperature (RT). The wells were then incubated with serially diluted antiserum or hybridoma culture supernatant for 90 min at 37°C, followed by goat anti-mouse IgG labeled with horseradish peroxidase (HRP) (whole IgG, diluted 1:1,000; Cappel, West Chester, PA, USA) for 60 min at 37°C. The amount of enzyme conjugate bound to each well was measured using 3,3',5,5'-tetramethylbenzidine (F. Hoffmann-La Roche Ltd., Basel, Switzerland) as a substrate, and the absorbance at 450 nm was read with an automatic ELISA analyzer (ImmunoMini NJ-2300; Nalje Nunc Int. Co. Ltd., Tokyo, Japan).
3
BrdU Proliferation Assay for HUVECs
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The bromodeoxyuridine (BrdU; (Roche, Basel, Switzerland) assay was performed as described previously (15 (link)). HUVECs incubated with different types of HDL at 100 µg/ml apoA-I concentration for 12, 24, 36 or 48 h. The cells were labeled with BrdU labeling solution (Roche) and then fixed with paraformaldehyde (Sigma-Aldrich). Following incubation with peroxidase-conjugated anti-BrdU working solution (Roche) for 90 min at 37°C, the cells were washed with washing buffer three times and substrate solution, 3,3′,5,5′-tetramethylbenzidine (Roche) was added. The absorbance of each well was measured at a wavelength of 450 nm with an ELISA plate reader (Model 550; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
4
ELISA Assay for Specific IgG Response
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Antibody (IgG) response of immunized mice was analyzed by ELISA assay. Briefly, purified rNS3 (3 μg/ml) was used as a captured molecule to coat 96-well polyvinyl chloride ELISA plates (Nunc, Denmark) for overnight at 4°C. After washing and blocking steps, wells were probed with optimum dilution of mice sera (1/1000 for total IgG), incubated for 1 hr, washed and further incubated with HRP-conjugated goat anti-mouse IgG (Sigma, Aldrich) as secondary antibody. Finally, by addition of TMB (Tetramethylbenzidine; Roche), color development was measured at 450 nm. IgG antibody subclasses were analyzed as mentioned above using goat anti-mouse IgG1, IgG2a, IgG2b antibodies (Sigma, Aldrich) and rabbit anti-goat IgG-HRP conjugate (Sigma, Aldrich). Optimum dilutions of mice sera were determined to achieve measurable ELISA signals against the coated antigen 27 (link).
5
ELISA-Based Murine IL-1beta Binding Analysis
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Example 6
The binding analysis was carried out using an enzyme-linked immunosorbent assay (ELISA)-based technology. The antigen murine IL-1beta (Sino Biologics, Cat. No. 50101-MNAE) was immobilized at a concentration of 0.5 μg/mL in 25 μL in PBS on a 384 well microtiter plate (Thermo Scientific, Cat. No. 464718). Every of the following steps was followed by a washing routine of 3 times 90 μL PBS, 0.5% BSA, 0.05% Tween with dispense and aspiration: 1) blocking step: saturating unbound surface (1 hour, 2% BSA); 2) anti-IL-1beta antibody in increasing concentrations for 1 hour; 3) detection antibody, dilution=1:2000 (Donkey F(ab)2 anti-rabbit IgG POD, Amersham, NA9340V or sheep IgG anti-mouse IgG POD, Amersham RPN4201). 20-30 min. after adding the substrate 3,3′,5,5′-tetramethylbenzidine (TMB, Roche Diagnostics GmbH, Mannheim, Germany, Cat. No 11835033001) the optical density was determined at 370 nm. The EC50 was calculated with a four parameter logistic model using GraphPad Prism 6.0 software.
6
IL-1beta Binding Assay Protocol
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Example 5
The binding analysis was carried out using an enzyme-linked immunosorbent assay (ELISA)-based technology. The antigen human IL-1beta (Sino Biologics, Cat. No. 90010CNAE) was immobilized at a concentration of 0.5 μg/mL in 25 μL in PBS on a 384 well microtiter plate (Thermo Scientific, Cat. No. 464718). Every of the following steps was followed by a washing routine of 3 times 90 μL PBS, 0.5% BSA, 0.05% Tween with dispense and aspiration: 1) blocking step: saturating unbound surface (1 hour, 2% BSA); 2) anti-IL-1beta antibody in increasing concentrations for 1 hour; 3) detection antibody, dilution=1:2000 (Donkey F(ab)2 anti-rabbit IgG POD, Amersham, NA9340V or sheep IgG anti-mouse IgG POD, Amersham RPN4201). 20-30 min. after adding the substrate 3,3′,5,5′-tetramethylbenzidine (TMB, Roche Diagnostics GmbH, Mannheim, Germany, Cat. No 11835033001) the optical density was determined at 370 nm. The EC50 was calculated with a four parameter logistic model using GraphPad Prism 6.0 software.
7
Cell Proliferation ELISA for HCV NS3
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This assay was carried out using the cell proliferation ELISA, BrdU (5-bromo-2 deoxyuridine) kit (Roche, Germany) according to the manufacturer’s instructions. Briefly, the splenocytes (3×105 cells/well) were co-cultured with 10 μM of rNS3-HCV in a 96-well culture plate (Nunc, Denmark) for 60 hr at 37°C and 5% CO2 and further incubated with BrdU solution (10 μM/well) for 12 hr. After washing and addition of FixDenat, the wells were probed with Anti Brdu –POD antibody for 90 min. Finally, proliferation analysis was measured by addition of TMB (Roche, Germany) at O.D. of 490 nm. Stimulation Index (SI) was calculated from the absorbance of stimulated/un-stimulated blanks.
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This assay was carried out using the cell proliferation ELISA, BrdU (5-bromo-2 deoxyuridine) kit (Roche, Germany) according to the manufacturer’s instructions. Briefly, the splenocytes (3×105 cells/well) were co-cultured with 10 μM of rNS3-HCV in a 96-well culture plate (Nunc, Denmark) for 60 hr at 37°C and 5% CO2 and further incubated with BrdU solution (10 μM/well) for 12 hr. After washing and addition of FixDenat, the wells were probed with Anti Brdu –POD antibody for 90 min. Finally, proliferation analysis was measured by addition of TMB (Roche, Germany) at O.D. of 490 nm. Stimulation Index (SI) was calculated from the absorbance of stimulated/un-stimulated blanks.
8
Measuring PMIF-CD74 Interaction in Malaria Sera
9
Protein Quantification via Western Blot
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The siRNA-transfected cells were harvested after 72 h and were washed twice with PBS. 1-2 Â 10 5 cells in a volume of 200 ml PBS were directly dissolved in 40 ml of 6x sample buffer (Mercaptoethanol, 6% SDS, 9% 2-375 mM Tris-HCl pH 6.8, 48% glycerol, and 0.03% bromophenol blue) and incubated for 5 min at 95 C. 20 ml of each of the samples were loaded in each of the wells of a 15% SDS-PAGE gel. After electrophoresis at 120 mv for 1.5 h, the separated proteins were electrotransferred to a PVDF nitrocellulose membrane (Roche). Blocking was performed using a 1x Roche blocking buffer dissolved in PBS over a 4-h period at room temperature. The membrane was incubated with primary antibodies diluted 1/200 in TBST buffer and left overnight at a temperature of 8 C. Anti-p53 (2B2.68), anti-HPV16 E6 (C1P5), anti-HPV16 E7, RB (Rb1), FKHR (C-9), goat anti-mouse IgG-HRP, p21 (F-5) and GAPDH (A-3) monoclonal antibodies were obtained from Santa Cruz Biotechnology Incorporation (Santa Cruz, CA). After washing with TBST buffer, the membrane was incubated with HRP secondary antibody in a dilution of 1/5000 for 1 h at 37 C. The membrane was washed three times with TBS buffer and incubated with TMB from Roche for 10 min at 37 C in a dark place. Western blot bands were quantified by imageJ software.
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